Optimization Of Screening, Identification And Fermentation Conditions On Amylase-producing And Protease-producing Strains In Pig Manure

With the expansion of aquaculture and increase of intensive level, the pollution caused by farm waste problem is getting worse, including animal manure pollution and ozone pollution. At present, the solution to the problem of pollution, including two kinds: one is the feces after treatment, mainly by composting, by adding a number of adsorbent, microbial agents, to make solid feces harmless and resources as soon as possible, and to reduce the ozone pollution; the other is the source from the feces of livestock after treatment, mainly through the feeding of probiotics and enzymes, to improve animal digestibility, to reduce the harmful microorganisms colonization in the gastrointestinal tract and to reduce the H2S, NH3, volatile fatty acids and other major components of ozone generation.This study mainly from animal manure and compost products using NH3 selective media preliminarily to screen bacteria total of 17 strains that can use ammonia, and quantitatively analysed 17 strains of this bacteria ability to produce amylase and protease, from which selected 4 high activity strains that could use NH3, using 16SrRNA Identification and molecular biology combine traditional biochemical identification methods, these 4 strains are:Bacillus subtilis, Bacillus stearothermophilus, Lactobacillus acidophilus, Enterococcus faecium. And we studied the physiological characteristics of the 4 strains, including resistance to bile salts and acid, acid production, dynamic growth curve. Among them, these four strains could survive in the gastrointestinal tract and maintain a certain vitality in the small intestine of pigs of bile salt concentration from 0.03% to 0.3%, and their ability of acid production was good, when training Lactobacillus acidophilus for 48h, pH 4.73.The study adopted Single-factor experimental design, Plackett-Burman design and central composite design (CCD) of Bacillus subtilis, Bacillus stearothermophilus and Lactobacillus acidophilus in the liquid fermentation conditions for optimization to selecte significant factors impacting three bacteria, and analyzed the significant factors by response surface design to determine the optimum level of each factor, and came to a conclusion by validated test. The optimal medium and culture conditions of the strains are as follows:(1) Bacillus subtilis:corn flour 1.607%, tryptone 1.977%, MgSO4·7H2O 0.0183%, MnSO4·H2O 0.102%,37℃, initial pH 7.0; final validation test for the determination of viable count 7.20×109, the theoretical value of 99.6%; (2) Bacillus stear other mophilus:1.534% of corn flour, soybean powder,3.169%, MgSO4·7H2O 0.331%, MnSO4·H2O 0.219%,37℃, initial pH 7.0; final validation number of viable cells measured 5.90×109,99.5% of the theoretical value;(3) Lactobacillus acidophilus:glucose 1.681%,2.2026% wheat bran, peptone 2.098%, K2HPO4 2.221%, Tween 80 1.152%,37℃, initial pH 6.5; final validation the number of viable cells measured 9.10×109,99.8% of the theoretical value.Based on optimized medium conditions, expand cultivating Bacillus subtilis and Lactobacillus acidophilus to the 5L fermenter to obtain a higher number of viable cells, respectively increased 1.5 and 2.1 times than before.